Electron microscopy of rapidly frozen lungs: evaluation on the basis of standard criteria

In recent years there has been a debate about the validity of the various methods for fixing lung tissue for electron microscopy in a state that faithfully reflects the physiological conditions prevailing at the time of fixation. Mazzone et al. (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 45: 325-333, 1978) introduced a method of rapid freezing followed by freeze-substitution fixation and found good preservation of fine structure; they claimed this method to be superior to others because it allowed careful control of physiological conditions. We have tested the suitability of this method for morphometric studies, where random sampling requires homogeneity of tissue preservation. The results are discussed on the basis of some standard criteria for the faithfulness of structural preservation. In terms of external standards, it was confirmed that one can find parts of the lung tissue samples to show a picture that is compatible with the established equivalent image of eucaryotic cells and tissues; however, the structure of blood was poorly preserved. In terms of the internal standards the method was found to yield inconsistent results; the specimens showed a wide spectrum of images of alveolar septa and capillaries, with "good preservation" limited to a very narrow area; furthermore, the method has a low level of reproducibility. We conclude that for the lung the method of freeze-substitution fixation is not suitable for morphometric work.